NAD+ and its reduced form NADH are essential cofactors in biological systems. They function as cofactors in over 300 redox reactions in vivo1. The level of NAD+ and NADH is tightly controlled by a variety of mechanisms including their biosynthesis and salvage. Therefore, it is difficult to answer some fundamental questions such as the minimal NAD+ level for cell growth and the biological consequences of abnormal activity of a specific NAD+-dependent enzyme. We expressed the NTT4 gene from Chlamydiae UWE25 in Escherichia coli BW25113, as the NTT4 protein was reported as a NAD+ transporter that can specifically transport intact NAD+ across cytoplasmic membrane2. We knocked out the nadC gene responsible for de novo biosynthesis of NAD+ and constructed the strain E. coli BW25113 (ΔnadC, NTT4). It was found that NAD+ in the culture media could significantly promote the growth of BW25113 (ΔnadC, NTT4), suggesting that the NTT4 protein was functional in E. coli (Fig A, B). We then disrupted the other two genes, nadD and nadE, and obtained the strains E. coli BW25113 (ΔnadD, NTT4) and BW25113 (ΔnadE, NTT4). Cell growth of these two strains are depending on exogenous NAD+ supplemented in the media, suggesting that we have successfully engineered E. coli to hold an NAD+ auxotrophic phenotype. We are carrying out a number of experiments using these NAD+ auxotrophic strains to address some interesting questions which may not be able to do otherwise. Results will be discussed during the conference.; NAD+ and its reduced form NADH are essential cofactors in biological systems. They function as cofactors in over 300 redox reactions in vivo1. The level of NAD+ and NADH is tightly controlled by a variety of mechanisms including their biosynthesis and salvage. Therefore, it is difficult to answer some fundamental questions such as the minimal NAD+ level for cell growth and the biological consequences of abnormal activity of a specific NAD+-dependent enzyme. We expressed the NTT4 gene from Chlamydiae UWE25 in Escherichia coli BW25113, as the NTT4 protein was reported as a NAD+ transporter that can specifically transport intact NAD+ across cytoplasmic membrane2. We knocked out the nadC gene responsible for de novo biosynthesis of NAD+ and constructed the strain E. coli BW25113 (ΔnadC, NTT4). It was found that NAD+ in the culture media could significantly promote the growth of BW25113 (ΔnadC, NTT4), suggesting that the NTT4 protein was functional in E. coli (Fig A, B). We then disrupted the other two genes, nadD and nadE, and obtained the strains E. coli BW25113 (ΔnadD, NTT4) and BW25113 (ΔnadE, NTT4). Cell growth of these two strains are depending on exogenous NAD+ supplemented in the media, suggesting that we have successfully engineered E. coli to hold an NAD+ auxotrophic phenotype. We are carrying out a number of experiments using these NAD+ auxotrophic strains to address some interesting questions which may not be able to do otherwise. Results will be discussed during the conference.